Aging, Plasminogen Activator Inhibitor 1, Brain Cell Senescence, and Alzheimer’s Disease

The etiology for late-onset Alzheimer’s disease (LOAD), which accounts for >95% of Alzheimer’s disease (AD) cases, is unknown. Emerging evidence suggests that cellular senescence contributes importantly to AD pathophysiology, although the mechanisms underlying brain cell senescence and by which senescent cells promote neuro-pathophysiology remain unclear. In this study we show for the first time that the expression of plasminogen activator inhibitor 1 (PAI-1), a serine protease inhibitor, is increased, in correlation with the increased expression of cell cycle repressors p53 and p21, in the hippocampus/cortex of senescence accelerated mouse prone 8 (SAMP8) mice and LOAD patients. Double immunostaining results show that astrocytes in the brain of LOAD patients and SAMP8 mice express higher levels of senescent markers and PAI-1, compared to astrocytes in the corresponding controls. In vitro studies further show that overexpression of PAI-1 alone, intracellularly or extracellularly, induced senescence, whereas inhibition or silencing PAI-1 attenuated H2O2-induced senescence, in primary mouse and human astrocytes. Treatment with the conditional medium (CM) from senescent astrocytes induced neuron apoptosis. Importantly, the PAI-1 deficient CM from senescent astrocytes that overexpress a secretion deficient PAI-1 (sdPAI-1) has significantly reduced effect on neurons, compared to the PAI-1 containing CM from senescent astrocytes overexpressing wild type PAI-1 (wtPAI-1), although sdPAI-1 and wtPAI-1 induce similar degree of astrocyte senescence. Together, our results suggest that increased PAI-1, intracellularly or extracellularly, may contribute to brain cell senescence in LOAD and that senescent astrocytes can induce neuron apoptosis through secreting pathologically active molecules, including PAI-1.

senescence contributes importantly to the pathophysiology of aging and aging-related diseases [1][2][3], including AD [4][5][6][7]. Senescent cells have been detected in the brain of AD patients [5][6][7][8] and AD model mice [4][5][6]. Removal of senescent cells pharmacologically or genetically alleviated brain A accumulation and tauopathy, two pathological features of AD, and improved memory in AD model mice [4][5][6], suggesting that cell senescence contributes importantly to the neuropathophysiology of AD. Nonetheless, what triggers brain cell senescence during aging and in AD, and how cell senescence leads to the pathophysiology of AD remain largely unknown. Finding answers to these questions will not only help understanding the mechanisms underlying AD but may also lead to identification of novel therapeutic targets for the treatment of this devastating disease.
Plasminogen activator inhibitor 1 (PAI-1), a primary inhibitor of tissue-type and urokinase-type plasminogen activators (tPA and uPA), plays a central role in hemostasis. Studies from this lab and others have shown that PAI-1 expression increases with age in the plasma [9,10] as well as in the brain of AD patients [11][12][13][14] and AD model mice [4,11,15,16]. Inhibition of PAI-1 activity or ablation of PAI-1 reduced brain A load and improved memory in familial AD model mice [11,[15][16][17], suggesting an essential role of PAI-1 in the pathophysiology of familial AD. Whether and how increased PAI-1 contributes to the neuropathophysiology in LOAD, the etiology, and the mechanisms of which is clearly different from FAD, however, is unknown and warrant further investigation [18][19][20].
In this study, we tested a hypothesis that aging-related increase in PAI-1 contributes to brain cell senescence and thus the neuropathophysiology during aging and in LOAD, using senescence accelerated murine prone 8 (SAMP8) mice, which harbor many behavioral and histopathological signatures of AD [21][22][23][24][25][26][27], and brain tissue samples from LOAD patients and age-matched healthy controls.
Pharmacological and genetic approaches were used to modulate PAI-1 activity and expression to define the role of PAI-1 in astrocyte senescence as well as the contribution of astrocyte SASP in neuron apoptosis in vitro. Our results support the notion that increased PAI-1 plays a critical role in brain cells, especially astrocyte, senescence and senescent astrocytes secrete pathologically active molecules, including PAI-1, which promote neuron apoptosis.

Human samples
High-quality human brain samples from the dorsolateral prefrontal cortex were obtained at autopsy from the longitudinal cohorts of the Alzheimer's Disease Research Center (ADRC, P30 AG072972) at University of California at Davis. The subjects had been well characterized by clinical, neuroimaging, neuropsychiatric, and post-mortem neuropathological analyses as: 1) those diagnosed with LOAD with high AD neuropathological changes (Braak stage V or VI, CERAD score 2 or 3, Thal phase 4 or 5) according to the current NIA-AA guidelines [28,29]; 2) Age-matched healthy individuals with minimal cognitive complaints and low AD neuropathological change (Braak stage < II, CERAD score 0 or 1, Thal phase <2) ( Table 1) as well as no significant vascular, Tau, TDP-43, or α-synuclein pathology. The samples had been de-identified at UCD Center before being delivered to us to protect the privacy of the individuals.

Mice
Senescence accelerated mouse prone 8 (SAMP8) mice have been described in our previous works [30,31] and CD-1 mice were from Charles River. The 50% SAMP8 mice were re-derived from the SAMP8 and CD-1 mice as previously described [32]. Four and twelve 12-monthold SAMP8 and wild type (CD-1) mice were sacrificed, and the hippocampus and cortex were dissected and fixed in 10% formalin or frozen down immediately in liquid nitrogen. All procedures involving animals were approved by the Institutional Animal Care and Use Committees at the University of Alabama at Birmingham.

Astrocyte isolation
Primary mouse astrocytes were isolated from cerebral cortex of neonatal C57BL/6J wild type mice and PAI-1 deficient mice (The Jackson Laboratory, Bar Harbor, ME) as we have described previously [33]. Briefly, the mouse brains were dissected, and the meninges were carefully removed. A mechanical dissociation of the cortices was performed by passing the tissue through a 1 ml sterile pipette. The homogenate was filtered through 100 m cell strainer and then centrifuged at 800 x g for 5 min at 4°C. The collected cell mixture was cultured in T75 flasks (one brain per flask) in high glucose DMEM supplemented with 10% FBS, non-essential amino acids (1X), penicillin/streptomycin (1X), 16 mM HEPES and 2 mM glutamine. After 2-week culture, the flasks were shaking on an orbital shaker at ~250 rpm for 5 hours to remove the microglial cells. The remaining astrocytes were trypsinized for further experiments.

Cell culture and treatment
Primary human astrocytes (ScienCell Research Laboratories, Catlog No #1800, Carlsbad, CA USA), cultured with Astrocyte Medium (Catlog No #1801, Carlsbad, CA USA), were transfected with non-targeted (NT) siRNA or PAI-1 siRNA (Santa Cruz, SC-37007 and SC-36179) for 24 hours and then treated with 200 µM H2O2 in serum-free medium for 2 days. Alternatively, the cells were infected with control and wild type PAI-1 expressing lentivirus overnight and then treated with 200 M H2O2 in the culture medium with no FBS nor virus for 2 days.
U87 MG human glioblastoma cell line was obtained from the American Type Culture Collection (Manassas, VA USA) and cultured with DMEM medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 g/mL streptomycin at 5% CO2 and 37 °C. U87 cells were transfected with non-target siRNA or PAI-1siRNA as described above and then treated with 200 M H2O2 for 2 days. Alternatively, the cells were treated with H2O2 in the presence or absence of 25 M TM5275 for 2 days.
Primary mouse cortical neurons were obtained from Thermo Fisher Scientific (Waltham, MA USA) and cultured with Neurobasal Medium supplemented with 0.2mM GlutaMAX-I Supplement, 2% B-27 Supplement, 100 units/mL penicillin, and 100 µg/mL streptomycin at 5% CO2 and 37°C. For the treatment of neurons with the conditional medium (CM) from senescent astrocytes, half of the medium in each well was replaced with the CM. The neurons were cultured with the CM for 2 days.

Immunofluorescence staining
To identify senescent astrocytes in SAMP8 mouse brain, double immunostaining was conducted using formalinfixed tissue slides with rabbit monoclonal antibody to MacroH2A1. 1  . Images were taken with a Nikon Andor Clara camera (Nikon). Over 300 astrocytes cells were counted in 6-9 different areas per mouse/human brain tissue; MacroH2A-, p16-, or PAI-1-positive astrocytes were counted, and the results were expressed as percentages of total astrocytes.

Measurement of the activity of senescence associated beta-galactosidase (SA--gal)
The activity of SA-β-gal in cultured cells was determined using 5-bromo-4-chloro-3-indolyl P3-D-galactoside (Xgal), following the protocol we have described previously [34]. SA--gal positive cells (blue color) were counted under microscope and expressed as percentages of total cells.

Analysis of Apoptotic neurons
Neuron apoptosis was assessed by three techniques: 1) Western analysis of apoptosis markers cleaved caspase 3 and Bax.
2) The activities of Caspase 3/7 and caspase 9: The activities of caspase 3/7 and caspase 9 in the neuron culture medium were assessed using assay kits from Promega (Madison, WI; Caspase-Glo 3/7 Assay) according to the manufacturer's protocols. 3) TUNEL staining techniques were used to detect apoptotic mouse neurons, using a kit from Sigma (Roche, Sigma 11684795910). Briefly, the cortical neurons (4x10 4 per well) were plated on 8-chamber slide (Thermo Scientific™ 154534) coated with poly-L-lysine (Gibco A38904-01). After cultured with the CM for 48 hours, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then washed with phosphate buffered saline (PBS) 3 times. The cells were permeabilized with Triton® X-100 and incubated with TUNEL staining reaction mixture (Roche, Sigma 11684795910). Fluorescence images were taken at UAB High resolution Image Facility and apoptotic neurons were counted manually in multiple areas for each well. The results are expressed as percentage of apoptotic neurons.

Statistical analysis
All of the "n" numbers presented in the Figure legends are the numbers of individual person/mouse/treatment, not replication of the same samples. Multiple group data were evaluated by one-way ANOVA while two group data were evaluated by t-test. Normality tests were conducted with Shapiro-Wilk program before group comparison and passed for all of the data, except the data presented in Fig. 4C, D, O and Fig 5H. Following oneway ANOVA, All Pairwise Multiple Comparison (Fisher LSD) was conducted to determine the significance of the difference between any two groups. For the data presented in Fig. 4C, D, O and Fig. 5H, which failed normality tests, the statistical analyses were conducted using Variance on Ranks, followed by Student-Newman-Kauls test. Correlation studies were performed with Pearson Product Moment Correlation program. Statistical significance was determined post-hoc by Tukey's test.

Correlated increases in PAI-1 and cell cycle repressors in the hippocampus/cortex of senescence accelerated mouse prone 8 (SAMP8) mice
SAMP8 mice are well-characterized murine model of accelerated aging, which harbor many behavioral and histopathological signatures of AD, including A deposition, tauopathy [31,35,36], and memory deficits [26,31,[35][36][37], and have been widely used to study the aging-related neuropathophysiology and screen candidate drugs for AD [26,31,[35][36][37]. Despite intensive characterization, the genes responsible for the accelerated senescence phenotype and neuropathological changes in SAMP8 mice remain unclear. To explore the mechanism underlying the neuropathological changes and memory loss in SAMP8 mice, the expression of PAI-1 protein and cell cycle repressors p53 and p21 were assessed by Westerns. The results show that PAI-1 protein is significantly increased, which is correlated with the increases in p53 and p21 in the hippocampus/cortex of 12month-old SAMP8 mice, compared to age-matched WT mice (Fig. 1A, B). Pearson Product Moment Correlation studies indicate that the amount of PAI-1 protein is closely correlated with the amounts of p53 (R=0.930, p<0.0001) and p21 (R=0.934, p<0.0001) (Fig. 1C). We also found that the increase of PAI-1 in the hippocampus/cortex of SAMP8 mice is age-dependent and so are the increases of p53 and p21 proteins (Fig. 1D-F). Immunofluorescence staining further show that the numbers of senescent astrocytes, which are stained positively with antibodies to macroH2A1a1 (a cell senescence marker) and GFAP (an astrocyte marker), are significantly increased with increased age in the hippocampus (Fig. 1G, H) and cortex of SAMP8 mice (Fig. 1G, I). Together, our data suggest that increased PAI-1 may underlie brain cell senescence, including astrocyte senescence, in SAMP8 mice.

PAI-1 protein level is increased in correlation with increased expression of cell cycle repressors p53 and p21 in the prefrontal cortex of LOAD patients
Senescent cells, including astrocytes, microglia, endothelial cells, and neurons have been detected in the brain of AD patients. To define the role of PAI-1 in brain cell senescence in AD, we assessed the expression of PAI-1 as well as cell cycle repressor p53 in the dorsolateral prefrontal cortex of LOAD patients and age-matched healthy controls by Westerns. The results show that PAI-1 protein level is significantly increased in the dorsolateral prefrontal cortex of LOAD patients, relative to that in agematched healthy controls ( Fig. 2A, B). This is associated with increases in the expression of p53 and p21 ( Fig. 2A,   B). Pearson correlation studies show that the amounts of PAI-1 proteins in individual subjects are positively correlated with the amounts of p53 proteins (Correlation Coefficient 0.618, p<0.0141) and p21 protein (Correlation Coefficient 0.745, p<0.001) (Fig. 2C).
Double immunostaining further showed that the numbers of PAI-1 positive astrocytes (Fig. 2D, E) and p16 positive astrocytes (Fig. 2F, G) are significantly higher in the dorsolateral prefrontal cortex of LOAD patients compared to that in age-matched healthy controls. These data suggest that more cells, including astrocytes, in LOAD patients compared to healthy controls, undergo senescence and that the increased PAI-1 expression may play a role in brain cell senescence in LOAD.

PAI-1 mediates H2O2-induced senescence in human astrocyte cell line U87 cells
To define the role of increased PAI-1 in astrocyte senescence, U87 cells, a human astrocyte cell line derived from malignant gliomas, were transfected with PAI-1 siRNA or non-targeted siRNA (NT-siRNA) and then treated with 200  HO. The results show that treatment of U87 cells with H2O2 increased PAI-1 protein levels in the cells and in the medium (Fig. 3A-C). H2O2 treatment also induced p21 and suppressed phosphorylation of retinoblastoma (pRb) (Fig. 3A, B), which was associated with an increase in the activity of senescence associated beta galactoses (SA--gal) (Fig.  3D, E), indicating that H2O2 induces U87 cell senescence. Silencing PAI-1, on the other hand, abrogated H2O2induced p21 expression and Rb dephosphorylation (Fig.  3A-B). This was associated with a dramatic reduction of SA--gal activity (Fig. 3D, E), suggesting that increased PAI-1 mediate H2O2-induced U87 cell senescence. Moreover, we show that treatment with TM5275, a small molecule PAI-1 inhibitor, almost completely blocked H2O2-induced p53 and p21 expression (Fig. 3F, G) as well as SA--gal activity (Fig. 3H, I) in U87 cells. The data further support the notion that increased PAI-1 mediates H2O2-induced U87 cell senescence.

PAI-1 mediates H2O2-induced senescence in primary human and mouse astrocytes
To further confirm involvement of PAI-1 in astrocyte senescence, primary human astrocytes, passages 3-5, were treated with 200 M H2O2 or transfected with PAI-1siRNA/NTsiRNA and then treated with H2O2. The results show that treatment with H2O2 induced PAI-1, p53, and p21 ( Fig. 4A-D) and increased the activity of SA--gal (Fig. 4E, F). Silencing PAI-1, on the other hand, significantly reduced H2O2-induced p53 and p21 expression as well as SA--gal activity (Fig. 4A-F), suggesting that H2O2 induces primary human astrocyte senescence through a PAI-1 dependent mechanism. We also show that transduction of astrocytes with lentivirus expressing wild type of PAI-1 increased the amount of matured form of PAI-1 protein (MW 42kd), although it had no significant effect on the expression of the precursor form (MW 45kd) (Fig. 4G-I). H2O2 treatment, on the other hand, increased the amounts of both precursor form and matured form of PAI-1 proteins (Fig. 4G-I). Importantly, overexpression of PAI-1 alone increased p21 protein as well as SA--gal activity and enhanced H2O2induced p21 expression, although it had no further effect on H2O2-induced SA--gal activity (Fig. 4G-L). Moreover, we show that ablation of PAI-1 in astrocytes (isolated from PAI-1 -/mice) completely abolished H2O2induced p16 expression and significantly attenuated H2O2-induced pRb dephosphorylation (Fig. 4M-P) as well as IL-6 and IGFBP3 secretion (Fig. 4Q, R). Together, our data suggest that PAI-1 mediates H2O2-induced senescence in primary human and mouse astrocytes.

Astrocyte SASP promotes apoptotic responses in neurons and SASP PAI-1 mediates part of the effects
To explore the potential mechanisms by which senescent astrocytes contribute to AD neuropathology, SH-SY5Y cells, a neuroblastoma cell line, were cultured with the conditional medium (CM) from senescent U87 cells for 48 hours (Fig. 6A Flow chart). The results show that treatment with H2O2 induced U87 senescence (Fig. 6B-D) but had no significant effect on the activity of the caspase 3/7 (Fig. 6E), suggesting that H2O2 induces U87 senescence but not apoptosis. Importantly, we show that treatment with the CM (no H2O2 present, data not shown) from senescent U87 cells increased the expression of Bax and cleaved caspase 3 (Fig. 6F-H) as well as the activity of caspas3/7 (Fig. 6I) in SH-SY5Y cells, suggesting that U87 SASP promotes SH-SY5Y cell apoptosis.
To further investigate the role of SASP PAI-1 in astrocyte SASP-induced neuronal apoptosis, primary mouse cortical neurons were cultured with the CM from primary mouse astrocytes, which were isolated from PAI-1 deficient mice and transduced with control lentivirus, lentivirus expressing wild type PAI-1 (wtPAI-1) or secretion deficient PAI-1 (sdPAI-1). The CM did not contain viruses as the viruses were removed after overnight infection and the cells were cultured with viruses-free medium for 48 hours before the CM was collected. The results show that treatment of neurons with the CM from wtPAI-1 virus-infected PAI-1 -/astrocytes led to significant increases in the number of TUNEL positive cells (Fig. 6J, K) as well as the activities of caspas3/7 and caspase 9 (Fig. 6L, M), compared to neurons treated with the CM from virus vector-transduced PAI-1 -/astrocytes. Neurons treated with the CM from sdPAI-1 virus-infected PAI-1 -/astrocytes also showed a significant increase in the number of TUNEL positive cells, although the increase was not as dramatic as wtPAI-1 CM treated neurons (Fig. 6J, K). Treatment of mouse neurons with the CM from sdPAI-1 virus-infected PAI-1 -/astrocytes, however, did not significantly increase the activity of the caspas3/7 or caspase 9, although treatment of neurons treated with the CM from wtPAI-1-infected PAI-1 -/astrocyte did (Fig. 6L, M). As both wtPAI-1 and sdPAI-1 induced astrocyte senescence (Fig. 5) and as no PAI-1 was present in the CM of sdPAI-1 virus-infected PAI-1 -/astrocytes (Fig. 5A), our results indicate that SASP PAI-1 plays a role in astrocyte SASP-induced neuron apoptosis.

DISCUSSION
It has been well documented that the number of senescent cells increases with age and in aging-related diseases, including AD [1,[3][4][5][6][7][8][38][39][40]. The mechanisms underlying cellular senescence during aging and in agingrelated diseases, however, remain poorly understood. In this study, we show, for the first time, that the expression of PAI-1, a serine protease inhibitor that plays a central role in hemostasis, is significantly increased in the hippocampus/cortex and astrocytes in SAMP8 mice, an accelerated murine aging model, and in LOAD patients. This is correlated with the increases in the expression of cell cycle repressors p53, p21, and/or p16. Using primary human and mouse astrocytes, we further show that overexpression of PAI-1 alone intracellularly or extracellularly induced astrocyte senescence, whereas silencing or deletion of PAI-1 attenuated H2O2-induced p53/p21/p16 expression as well as astrocyte senescence. Moreover, we show that treatment of neurons with the conditional medium (CM) from senescent astrocytes induced neuron apoptosis whereas PAI-1 deficient CM had significantly less effect on neurons. As PAI-1 expression increases with age [9,41] and in AD [11][12][13], our results suggest that increased PAI-1 may underline brain cell senescence and neuropathology during aging and in LOAD (Fig. 7).
Increased PAI-1 has been used as a marker of cell senescence and has been shown to mediate cell senescence in vitro and in vivo [34,[42][43][44][45][46][47]. Eren et al. showed that PAI-1 expression was increased in Klotho mice, another accelerated murine aging model [45]. Deletion of the PAI-1 gene in Klotho mice by crossing these mice with PAI-1 knockout mice reduced plasma levels of insulin-like growth factor binding protein 3 (IGFBP3) and IL-6, two of cell senescence markers and mediators [48]. Using a secretome proteomics approach, Elzi et al. identified IGFBP3 as a secreted mediator of breast cancer senescence upon chemotherapeutic drug treatment [43]. They further showed that the senescenceinducing activity of IGFBP3 was inhibited by tPA, whereas PAI-1 stabilized IGFBP3 by inhibiting tPAmediated proteolysis of IGFBP3 [43]. Together, these data suggest that PAI-1 may mediate stress-induced cell senescence by increasing IGFBP3. Omer et al. reported, on the other hand, that sequestration of PAI-1 in stress granules (SGs) led to the translocation of cyclin D1 to nucleus and RB phosphorylation as well as suppression of senescence in two human diploid fibroblasts [46], suggesting that increased PAI-1 may promote cell senescence through modulating cyclin-pRb cell cycle repression pathway. In a previous study, we showed that specific ablation of PAI-1 in alveolar type II (ATII) cells in mice attenuated bleomycin-induced ATII cell senescence and lung fibrosis [34]. Using pharmacological and genetic approaches, we further showed that PAI-1 induced ATII cell senescence through activating p53-p21-pRb cell cycle repression pathways [34]. In contrast, we showed, in a different study, that PAI-1 mediated TGF-1-induced ATII cell senescence through inducing p16, not p53 [47]. In this study, we further show that H2O2 induced primary human and mouse astrocyte senescence through activating p53/p21/p16 cell cycle repressors. Although the underlying mechanism is still unknown, these data suggest that increased PAI-1 may promote brain cell senescence through increasing the expression of cell cycle repressors. Figure 7. Hypothetic mechanism by which PAI-1 promotes brain cell senescence and neuron apoptosis during aging and in LOAD. PAI-1 expression increases with age and in LOAD brain. Increased PAI-1, intracellular or extracellular, leads to increases in the expression of cell cycle repressors p53, p21, and/or p16 as well as senescence in brain cells, including astrocytes. Senescent astrocytes in turn secrete pathologically active molecules, including PAI-1, which induces neuron apoptosis.
PAI-1 is a secreted protein and is believed to function mainly in extracellular space, although a few studies suggest that intracellular PAI-1 may also have important functions [18][19][20]. To define the role of intracellular PAI-1 in cell senescence, we constructed a secretion deficient PAI-1 (sdPAI-1) expression lentivirus by deletion of the nucleotides coding the secretion signal peptides. We Aging and Disease • Volume 14, Number 2, April 2023 526 found that transduction of PAI-1 -/astrocytes with sdPAI-1 expression lentivirus alone induced astrocyte senescence (increased p16 expression and SA--gal activity), just like wild type PAI-1 (wtPAI-1) expression lentivirus did. These data suggest that an increase in the intracellular PAI-1 alone is sufficient to activate cell cycle repression pathway and induce senescence. Our data further support the notion that intracellular PAI-1 also has important functions, although the mechanism by which intracellular and extracellular PAI-1 activates cell cycle repression pathways remains to be further investigated. Senescence accelerated murine prone 8 (SAMP8) mice are a naturally occurring mouse line displaying a phenotype of accelerated aging [49,50]. The lifespan of SAMP8 mice is 40% shortened [21,50] and these mice harbor many behavioral and histopathological signatures of AD, including A deposition, tauopathy, and memory deficits [26,31,[35][36][37]. Although the accelerated aging and neuropathophysiology phenotypes have been well characterized in SAMP8 mice, the gene(s) that is (are) responsible for the phenotypes remain unclear. In this study, we show for the first time that PAI-1 expression is significantly increased in the hippocampus/cortex of SAMP8 mice, associated with increased expression of cell cycle repressors p53 and p21, suggesting that increased PAI-1 may underlie the aging phenotype. This notion is supported by the observations from Klotho mice, another murine aging model [45]. Eren et al. reported that PAI-1 expression has increased in Klotho mice. Deletion of the PAI-1 gene reversed senescence phenotype, preserved organ structure and function, and prolonged the lifespan in Klotho mice [45]. Most interestingly, Khan et al. reported that people at Berne Amish community who carry heterozygous mutation (c.699_700upTA) in the PAI-1 gene with a loss of function of the protein have significantly longer leukocyte telomere length, lower fasting insulin levels, and lower prevalence of diabetes mellitus whereas the carriers of the null PAI-1 allele had a longer life span [51]. These data further support the critical role of PAI-1 in aging processes in humans. Nonetheless, whether increased PAI-1 is responsible for the accelerated aging phenotype and neuropathophysiology in SAMP8 mice and in human population remains to be further investigated.
Although emerging evidence indicates that cell senescence plays a critical role in the pathophysiology of aging and aging-related diseases, including AD, the underlying mechanism remains unclear. In a previous study, we showed that treatment of alveolar macrophages with the conditional medium (CM) from senescent type II alveolar epithelial (ATII) cells stimulated the expression of the genes associated with a pro-fibrotic phenotype in alveolar macrophages [47]. Deletion or inhibition of PAI-1 attenuated TGF-1 induced ATII cell senescence, SASP, and the stimulatory effects of ATII cell SASP on macrophages [47]. These data support the notion that senescent cells secret biologically active molecules that adversely affect the survival or function of adjacent cells. Astrocytes, the most abundant cell type in the brain, are closely associated with neurons and are essential for neuron survival and function. In this study, we show, for the first time, that the CM from senescent astrocytes promotes apoptotic responses in neurons. Importantly, the CM containing no PAI-1 had a significantly reduced capacity to induce neuron apoptosis compared to PAI-1 contained CM, although secretion deficient PAI-1 induced similar levels of astrocyte senescence as wild type PAI-1. These data suggest that senescent astrocytes can promote neuron apoptosis by secreting pathogenically active molecules including PAI-1.
More works are needed to understand how SASP PAI-1 promotes neuron apoptosis.
In summary, we show for the first time that PAI-1 expression is increased, in correlation with the increased expression of cell cycle repressors p53/p21/p16, in the hippocampus/cortex of SAMP8 mice and LOAD patients. In vitro studies further suggest that increased PAI-1 expression may underlie astrocyte senescence and that senescent astrocytes can promote neuron apoptosis by secreting pathologically active molecules, including PAI-1. More works are needed to elucidate the mechanism by which PAI-1 regulates the expression of cell cycle repressors in astrocytes and how SASP promotes neuron apoptosis. Whether increased PAI-1 is responsible for the aging phenotype in SAMP8 mice and in human being also warrants further investigation.